In recent years, many studies were carried out for developing new food packaging systems. There is a variety of synthetic and natural antimicrobial compounds used to control the growth of microorganisms. The use of natural antimicrobial agents, due to the low side effects and high antibacterial potential, has captured the attention of scientists. The main aim of this study was to produce polylactic acid-based biodegradable active films containing lemon verbena Essential oil nanoemulsion to control the growth of two common foodborne pathogenic bacteria (Staphylococcus aureus and Escherichia coli) in Rainbow Trout. To aim this, the antibacterial activity (in vitro) of the films were assessed under MIC and MBC assays using the microdilution method. The nanoemulsion was prepared by ultrasonic waves. Based on dynamic light scattering, the mean droplet size was reported at about 22.4 nm. Based on the antibacterial results, the growth of both microorganisms was significantly decreased after 0, 3, and 7 days of storage in comparison with the control group (p <0.05). To sum up, the presence of Lippia citriodora Nanoemulsion in the matrix of the polylactic acid film showed notable antibacterial activity during 7 days of storage at 4˚C (refrigerator). It can be concluded that this film can be a good candidate for food packaging purposes to control the growth of microorganisms.